Evidences of N2O emissions in chloropicrin-fumigated soil - Journal of

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Evidences of N2O emissions in chloropicrin-fumigated soil wensheng fang, Dongdong Yan, xianli wang, bin huang, Zhaoxin Song, jie liu, Xiaoman Liu, Qiuxia Wang, Yuan Li, canbin ouyang, and Aocheng Cao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04351 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

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Journal of Agricultural and Food Chemistry

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Evidences of N2O emissions in chloropicrin-fumigated soil

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Wensheng Fang, Dongdong Yan, Xianli Wang, Bin Huang, Zhaoxin Song, Jie Liu,

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Xiaoman Liu, Qiuxia Wang, Yuan Li, Canbin Ouyang, Aocheng Cao*

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Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193,

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China

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*Corresponding

author email address: [email protected], Tel: +86-62-815-904

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ACS Paragon Plus Environment


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Abstract

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The mechanism of N2O production following chloropicrin (CP) fumigation was

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investigated in this study. Our results showed that CP fumigation increased N2O production

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from 23 to 25 times compared with the control, and significantly decreased the abundance

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of 16S rRNA and N-cycling functional genes. CP also decreased the soil bacterial diversity

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and caused a shift in the community composition. The N2O emissions in fumigated soil

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were significantly correlated with soil environmental factors (NH4+, dissolved amino acid,

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microbial biomass nitrogen, and NO3−) but were not correlated with the abundance of

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functional genes. Metatranscriptomes and dual-label 15N-18O isotope analysis revealed that

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CP fumigation inhibited the expression of gene families involved in N2O production and

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sink processes and shifted the main pathway of N2O production from nitrification to

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denitrification. These results provided useful information for environmental safety

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assessments of CP in China, for improving our understanding of the N-cycling pathways

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in fumigated soils.

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Keywords: Chloropicrin, N2O emissions, Functional genes, Nitrogen cycling,

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Microorganisms

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Introduction

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Soil fumigation has been used to control soilborne pathogens, nematodes, and weeds

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for several decades worldwide. Methyl bromide (MeBr) was the most used and best

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fumigant for more than 50 years 1. However, the use of MeBr has been completely 2


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prohibited in agriculture system since 2015 because of its detrimental effect on ozone 2. As

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one of the most suitable alternatives to MeBr, chloropicrin (CP) does not deplete ozone

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and exhibits high efficacy in killing board-spectrum soil-borne diseases by rapidly

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immersing into the tissues of biota and killing cells 3. To date, CP has been widely

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employed in crops, such as strawberry, cucumber, tomato, ginger, melon, and eggplant, to

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control soil-borne pathogens and plant-parasitic nematodes 4-6.

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Soil fumigation can effectively target organisms but also affects off-target

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microorganisms, which are important to the soil microbial community. Studies reported

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that CP causes a significant decrease in bacterial diversity and shift on the population size

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and soil bacterial composition

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transforming activities, including mineralization stimulation 9 and nitrification reduction

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10,11.

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of microbial community in increasing N2O emissions and the biochemical pathways used

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by microorganisms to produce N2O emissions after CP fumigation have not been reported

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yet.

7,8.

CP fumigation triggers significant effects on N-

Soil treated with CP increased N2O emissions by 12.6 times 12,13. However, the role

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N2O is a potent greenhouse gas with a global warming potential that is 298-fold higher

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than that of CO2 14. N2O is one of the most significant ozone depletes that affect the long-

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term recovery of the ozone layer

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primary sources of N2O. Nitrification starts with the oxidation of NH3 to NO2− and of NO2−

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to NO3− 16. Under aerobic conditions, chemoautotrophs use NH3 as energy source 17,18 to

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produce N2O through several pathways, such as nitrifier nitrification (NN), nitrifier

15.

Microbial nitrification and denitrification are the

3



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denitrification (ND), and nitrification-coupled denitrification (NCD). The biological

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denitrification

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NO3−→NO2−→NO→N2O→N2; these steps are catalyzed by NO3− reductase (nar/nap),

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NO2− reductases (Nir), nitric oxide reductases (Nor), and N2O reductase (nos), respectively

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19.

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induced by N2O-reducing microorganisms indicates that they are the only microbial sink

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for N2O

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codenitrification

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lead to N2O formation.

process

consists

of

four

reaction

steps,

namely,

N2O can result from a multiplicity of pathways, but the process of N2O reduction to N2

20.

Other microbial processes, such as heterotrophic nitrification 22,

21,

chemodenitrification, and dissimilatory NO3− reduction to NH3, can

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In this study, we examined the production of N2O and the response of different groups

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of N-cycling microbes living in Jiangxi lateritic red and Beijing Fluvo-aquic soils

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fumigated with CP. At 59 days after fumigation, we quantified N2O emission rates, changes

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in geochemical parameters NO3− and NH4+, microbial biomass nitrogen (MBN), and

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dissolved amino acids (DAA). We also used real-time PCR to monitor the abundance of

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key functional marker genes (nifH, AOA amoA, AOB amoA, nxrB, narG, napA, nirK, nirS,

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cnorB, qnorB, and nosZ) involved in microbial N fixing, nitrification, and denitrification.

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Moreover, 16S rRNA gene amplicon sequencing techniques were used to determine the

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diversity and community structure of N-transfer microorganisms in the fumigated soils.

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Finally, we analyzed dual-labeled 15N-18O isotopes and metatranscriptome to observe shifts

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in the N2O pathway and determine the possible production mechanism of N2O in the

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fumigated soils. We aimed to (i) discuss the biochemical pathways used by microorganisms 4


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to produce N2O emissions from soils fumigated with CP and (ii) determine the response

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and recover rate of the N-cycling microorganisms in CP-fumigated soil at the post-

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fumigation phase. Results are predicted to improve the understanding of N-cycling

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mechanisms in fumigated soil ecosystems and provide users with valuable guidance

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regarding the efficient and effective management of CP.

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Methods and Materials

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Soil sample collection

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Soil samples were obtained from the top 20 cm of two agricultural fields in Beijing,

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Shunyi and Jiangxi, Yudu. The two samples are typical alkaline Fluvo-aquic (Beijing) and

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acidic lateritic red soils (Jiangxi). Detailed soil analytical data are presented in Table 1.

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The soils were preincubated for 7 days at room temperature (25 ± 5 °C) in the dark and

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then adjusted to 40% water-holding capacity (WHC) before fumigation.

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Experiment I: Post-fumigation soil incubation experiment

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Experimental setup

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Microcosms were prepared with 220 g of sieved soil in 500 mL Duran® wide-neck

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glass bottles (Schott AG, Mainz, Germany). CP (99.5% purity) was obtained from Dalian

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Dyestuffs & Chemicals Co., Dalian, Liaoning, China. CP was added at a typical field

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application rates of 65 mg kg−1 to both soil types as treatment groups. The control group

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was treated with deionized water. Each treatment was prepared triplicates. The soil

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microcosm bottles were closed with butyl rubber stoppers with outlet ports for syringe

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sampling and incubated at the ambient temperature of 28 °C in daylight. 5



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Soil sampling and geochemical analyses

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N2O emission rates were measured using the method previously described 14,23. During

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the incubation stage, the soil samples in the bottles were thoroughly stirred for 10-15 min

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every day to create an aerobic incubation environment. At the designated time, a 10 mL

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gas sample was withdrawn from each bottle using a gas-tight syringe (Agilent

111

Technologies, USA). The gas samples were transferred to a 21 mL headspace vial that was

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flushed with helium and evacuated (10 mL) before use. An Agilent 7890A gas

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chromatograph with an electron capture detector (63Ni-ECD) coupled with an Agilent

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7694E headspace sampler were used to quantify the N2O concentrations. The gas

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chromatograph conditions and gas emission calculation method were described previously

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24.

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any residual fumigant. After sampling, all bottles were pumped with fresh air and then

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returned into the incubator. During incubation, water content was controlled

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gravimetrically and adjusted each week to the initial WHC by adding deionized water by

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a spray bottle. Gas samples were collected every week, and soil samples were collected at

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days 10, 24, 38, and 59 for geochemical and molecular biological analyses.

After each gas sample, the microcosm bottles were opened in a ventilation hood to vent

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Soil samples were extracted for mineral N (NH4+-N and NO3–-N) by using 2 M KCl.

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Mineral N concentrations were determined with a continuous flow automated analyzer

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(Futura Continuous Flow Analytical System, Alliance Instruments, France). MBN was

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estimated by the chloroform fumigation method

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ninhydrin reaction method 26.

25,

and DAA was measured using the

6


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Real-time quantitative PCR and high-throughput sequencing

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Soil total genomic DNA was extracted from 0.25 g of each soil sample by using a

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MoBio Powersoil® DNA Isolation Kit (MoBio Laboratories, CA, USA) according to the

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manufacturer’s protocol. Quantification of functional marker genes (16S rRNA gene

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[bacteria], amoA [bacteria and archaea], nifH, napA, narG, nirK, nirS, cnorB, qnorB, and

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nosZ) was carried out using the SsoFast EvaGreen Supermix (Bio-Rad Laboratories, CA,

133

USA) and gene-specific primers. Quantitative PCR was performed using CFX96 Real-time

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PCR System (Bio-Rad, CA, USA). The details of gene-specific qPCR primers, reaction

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mixtures, and thermal programs are listed in Tables S1, S2, and S3, respectively. To

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analyze the abundance and community structure of N-transfer microbes, Majorbio Bio-

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pharm Technology Co., Ltd. (Shanghai, China) conducted MiSeq sequencing of the 16S

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rRNA genes in the V3–V4 regions by using the total DNA extracted from Beijing soil

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microorganisms. The raw reads were deposited into the NCBI Sequence Read Archive

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database (no. SRP124701). All the certified nitrogen cycle related functional microbes

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were collected from the NCBI database (https://www.ncbi.nlm.nih.gov/) (showed in Table

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S4 & Table S5).

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Experiment II: Short-term fumigation experiment

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Experimental setup

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A total of 3 g (dry weight) incubated Beijing soil samples (the same soils used in

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Experiment I) were placed into a 21 mL clean headspace vial. The following treatments

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for each fumigant were established: (1) 18O-H2O + NO3− + NH4+; (2) H2O + 18O-NO3− + 7



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NH4+; (3) H2O + 15N-NO3− + NH4+; and (4) H2O + NO3− + 15N-NH4+. The isotopes 18O and

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15N

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treatments received the total mineral N contents in the soil of 50 mg NH4+-N and 50 mg

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NO3−-N kg−1 soil. All treatments were incubated at 40% of WHC at 28 °C. Each treatment

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was prepared in triplicates for each isotope and soil total RNA extraction. The gas and soil

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samples were taken at the end of 70 h of incubation. Our preliminary experiment found

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that the volume of N2O emitted from the soil peaked at 70 h after fumigation.

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Isotope-labeled gas and soil sample analyses

(H2O, NO3−, NH4+) were enriched with 1.0%

18O

and 10.0%

15N,

respectively. All

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The rate of N2O emissions and its isotopic signatures were recorded at the Institute of

157

Environment and Sustainable Development in Agriculture (CAAS) by using an IsoPrime

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trace gas concentration system interfaced to an IsoPrime100 isotope ratio mass

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spectrometer (IsoPrime Ltd., UK). Soil samples, which were extracted with 1 M KCl

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followed by segmented flow analyses, were obtained after gas sampling (Skalar Analytical

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B.V., Breda, The Netherlands). The concentrations of the 15N enrichments were analyzed

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using an elemental analyzer interfaced to a continuous flow isotope ratio mass spectrometer

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(Sercon 20-20, Sercon Ltd., UK). The 18O and 15N isotopic enrichments of the accumulated

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N2O, the 18O signature in H2O and NO3−, and the 15N signatures in NO3− and NH4+ were

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used to calculate the N2O produced by NN, ND, NCD, and heterotrophic denitrification

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(HD) N2O pathways, respectively 27,28.

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Metatranscriptomic analysis

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Total RNAs were extracted from nine samples by using the EZNA® Soil RNA Midi 8


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Kit (Omega Bio-tek Inc., USA) according to manufacturer’s protocols. The RNA

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concentration

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spectrophotometer (Thermo Fisher Scientific, USA). RNA quality was examined using a

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1% agarose gel electrophoresis system and assessed using RNA 6000 Nano Kit and

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Reagents (total RNA) in an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The

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total RNA contained in samples was based on an rRNA removal procedure by using the

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Ribo-Zero rRNA Removal Kit (Illumina Inc., USA) according to the manufacturer’s

176

instructions. cDNA libraries were constructed using the TruSeq™ RNA Library Prep Kit

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v2 (Illumina Inc., USA) according to the manufacturer’s instructions. The barcoded

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libraries were paired end sequenced on the Illumina HiSeq 2500 System (Illumina Inc.,

179

USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) by using HiSeq

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3000/4000 PE Cluster Kits (Illumina Inc., USA) according to the manufacturer’s

181

instructions. A total of 325,931,416 raw reads were obtained. Quality control was carried

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out by the SeqPrep (https://github.com/jstjohn/SeqPrep) to strip the 3′ and 5′ ends, as well

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as by Sickle (https://github.com/najoshi/sickle) and SortMeRNA

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reads (length < 50 bp, with a quality value < 20, or possessing N bases) and rRNA reads,

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thereby resulting in 200,925,212 high-quality sequences. A total of 30,093 open reading

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frames with an average length 463 bp from each sample were predicted using

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TransGeneScan (http://sourceforge.net/projects/transgenescan/). All sequences with a 95%

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sequence identity (90% coverage) were clustered as nonredundant gene catalog by the

189

program CD-HIT (http://www.bioinformatics.org/cd-hit/). Reads after quality control were

and

purity

were

quantified

using

9


a

NanoDrop™

2000/2000c

to remove low-quality


190

mapped to the representative genes with 95% identity and reads per kilobase of transcript

191

per million mapped reads and evaluated using RNA-Seq by expectation maximization

192

(http://deweylab.biostat.wisc.edu/rsem/). Functional and taxonomic assignments of the

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predicted genes used the Basic Local Alignment Search Tool (BLAST) program to

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compare nucleotide or protein sequences contained in the databases eggNOG, KEGG, and

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NCBI nr. The BLAST E-value cut-off was set at 1.e−5. All metatranscriptomic data were

196

deposited in the NCBI database (no. SRP143409).

197

Statistical analysis

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The major effects of fumigation on the biochemical parameters and abundance of

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functional genes involved in N cycling was studied using univariate ANOVA with least

200

significant difference test by using the SPSS statistics software package version 18.0 (IBM,

201

USA). Univariate ANOVA was used to reveal differences between the control and

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fumigated microcosms. All concentrations or gene copy number values from the control at

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each time point of sampling were individually compared with the fumigated soil

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microcosms. The α-diversity indices Chao1, ACE, Shannon, and Simpson were calculated

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using the software Mothur to determine the diversity of the bacterial communities in

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Beijing soil. Hierarchical clustering and a heat map were used to determine changes in the

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relative abundance of the bacterial genera involved in N cycling. The Bray–Curtis

208

algorithm was used to show the relative abundance of these genera according to

209

hierarchical clustering. A heat map figure and statistical correlations were generated to

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show the sequencing results by using pheatmap-package and vegan package in R, 10


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respectively (version 2.15.3). Linear discriminant analysis coupled with effect size

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measurement was used to identify the relative abundance of the bacterial genera in CP-

213

fumigated and unfumigated Beijing soils. Spearman’s rank correlation coefficient was used

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to determine the relationship among N2O emission rate, physicochemical parameters, and

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microbial functional genes.

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Results

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N2O production rates

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The N2O production rates increased by 23.5- and 25.4-fold within 10 days of CP

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fumigation in Beijing and Jiangxi soil compared with unfumigated soils, respectively

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(Figure 1). However, N2O release rates gradually decreased at the post-fumigation phase

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and returned to emission levels similar to that of the control at days 45 and 38 in Beijing

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soil and Jiangxi soil.

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Changes in physiochemical parameters

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Compared with unfumigated soils, CP fumigation significantly increased the NH4+-N

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and DAA contents in both soil types, whereas NO3−-N and MBN concentrations were

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significantly decreased (Table 2). All these parameters recovered to levels similar to those

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of the controls 59 days after fumigation in Beijing soil. However, the NH4+-N, NO3--N, and

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MBN values were not recovered to the control levels until 59 days of fumigation in Jiangxi

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soil. This result suggested that CP induced larger changes in these parameters that persisted

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for longer in Jiangxi soil than in Beijing soil.

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Changes in abundance of 16S rRNA and N-cycling functional marker genes 11



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CP fumigation induced a significant decrease in 16S rRNA gene abundance in Beijing

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soil, and this inhibitory effect was evident in fumigated Jiangxi soil (Figure 2). The total

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bacterial abundance of Beijing soil following CP fumigation recovered to the control level

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38 days after treatment. However, in Jiangxi soil, CP fumigation inhibited the total

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bacterial abundance for the entire incubation period. The copy number of the observed 11

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N-cycling functional genes (nifH, AOA amoA, AOB amoA, nxrB, napA, narG, nirK, nirS,

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cnorB, qnorB, and nosZ) were significantly decreased in CP-fumigated Beijing and Jiangxi

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soils (Figure 2–4). In CP fumigated Jiangxi soil, only gene populations nxrB and nirS

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recovered to the control level 59 days after treatment. In CP-fumigated Beijing soil,

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functional genes, such as AOB amoA, nxrB, nirK, nirS, and cnorB recovered to levels

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similar to that of the control at day 38.

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Changes in soil bacterial diversity and community composition

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The Shannon, ACE, and Chao1 diversity indices decreased significantly (p < 0.01)

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relative to those in the control group in CP-fumigated Beijing soil samples during the entire

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incubation phase (Table 3), but the Simpson diversity index significantly increased (p
0.69, p < 0.0002) and DAA (r > 0.83, p

Evidences of N2O emissions in chloropicrin-fumigated soil - Journal of

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